RU2295980C1 - Implant for repairing bone and/or cartilage tissue and method for producing it - Google Patents

Abstract

FIELD: medical engineering.

SUBSTANCE: implant has osteocytes and/or chondrocytes in nutrient medium. The osteocytes and/or chondrocytes are obtained from fibroblasts of autologic marrow by culturing on nutrient medium comprising calf blood serum, collagen and growth factors, and, optionally, mucopolysaccharides. Osteocytes and/or chondrocytes content in implant makes 100000-100 millions of cells in 2-5 ml of the nutrient medium corresponding to conditions for culturing fibroblasts to produce osteocytes or chondrocytes. Method for producing implant involves homogenizing, centrifuging patient marrow. Deposit containing stromal fibroblasts is cultured on special nutrient medium. To achieve fibroblast specialization towards osteocytes, the fibroblasts are mainly cultivated on a nutrient medium containing calf blood serum, collagen of type I and type II, fibroblasts growth factor. To direct fibroblasts development specialization mainly in chondrocyte direction, the fibroblasts nutrient medium is applied, in which serum, collagen of type I and type II, and also mucopolysaccharides and insulin-like growth factor or Somatomedin hormone are available. Cultivation is carried out until 100 000 up to 1000 million cells are accumulated in the nutrient medium. Then, 2-5 ml of the nutrient medium is added corresponding to fibroblasts cultivation conditions to produce osteocytes or chondrocytes.

EFFECT: high level of biocompatibility; improved anti-inflammatory effect and comfort condition; reliable adhesion to bone tissue; minimum integration time, optimum osteochondrogenesis course in necrotized bones and degeneratively changed cartilage.

6 cl

Description

The invention relates to biology and medicine, in particular to a method for producing an autograft for restoration of bone and cartilage tissue, and can be used in rheumatology, traumatology, orthopedics, dentistry and cosmetology.

In the United States alone, approximately 1.3 million surgeries occur in bone implantation, bone substitution, or other reconstructive surgery. A large number of materials are known that are used as an implant or a substitute for bone substance. The implant material is bone meal (SU 1696818, RU 2189823), demineralized bone, collagen and fibroblasts (US 4485097), collagen and non-collagen proteins, polysaccharides, amino acids (US 5866165, RU 97116345, RU 95117043), placental, bone cell tissue and propolis (RU 21106781), embryonic chondrocytes or cells of germinal connective tissue (DE 3410631), also homopolymers and copolymers of polyglycolic and polylactic acids, copolymers of glycolide and lactide (WO 99/19005), glutaraldehyde (FR 2582517) and other materials including natural and synthetic polymer polymers. Ceramic implants, such as hydroxyapatite or tricalcium phosphate or tetracalcium phosphate (RU 2062622 C1, DE 3727606, FR 2585576) are another class of acceptable materials acceptable as implants.

However, the use of various natural and synthetic minerals, polymers and composites does not give the results that were achieved using autogenous materials. Approximately 90% of operations performed are related to autotransplantation, since alloprosthetics and xenoprosthetics entail a risk of pathogenic contamination and are accompanied by a long period of suppression of immunity. It is understood that the availability and availability of autotranspants is limited. The main limitations of this technology are taking material from the patient himself, preserving it in a physiological environment, the need for an additional healing process at the place of extraction of the material. In principle, this also applies to homologous allografts, i.e. donor transplants of the same species, in these cases there is also the problem of immunological compatibility and virus infection. Recent advances in cellular molecular biology and biotechnology of biomaterials have led to the development of tissue engineering, which allows constructing tissues using living cells, and developing methods for restoring functionally active tissues both in vitro and in vivo (US 6398816, WO 01/82773). Currently, a number of attempts are known based on the inclusion of isolated cells of biopsy origin, used as the basis for the creation of the implant. Cartilage cells (chondrocytes) and bone-forming cells (osteoblasts) continue to develop, combining with the existing structure of the patient’s bone or cartilage after administration, so that after a certain period of time, the implanted structure becomes completely indistinguishable from the surrounding tissue.

Cells of cartilage and bone, tendons and ligaments, and fibrous connective tissue are known to radically differ in character. An article by Alberts et al., Molecullar Biology of the cell., 2 Ed, 1989, p. 987-988 reports that bone matrix preparations can be implanted into the dermal layer of the skin, while they are transformed into cartilage cells and other bone cells tissue. US patent 57000289 describes the use of periosteum cells, which induce more bone cells than muscle cells. US 2003/0077825 A1 demonstrates the possibility of using fibroblasts of dermal, gingival or periodontal origin, taken from the patient himself, grown as cell bones, grown under cell growth conditions, in which the seeded cells differentiate into osteogenic type cells and then are incorporated into the structure of natural or synthetic gyroxyapatite or inorganic bone mineral. Patent EP 0339607 discloses a composition for regenerating articular cartilage and bone by implanting chondrocyte or osteoblast cells obtained from bone marrow of autologous or homologous origin or homologs including chondrocytes or autologous or homologous fibroblasts, obtained chondrocytes or any other progenital mesenital cells are placed in a biodegradable, biocompatible, immobilizing carrier. A portion of the implant may include a bone segment or bone substitute.

US patent 6596274, which we have taken as the closest analogue, describes biological material consisting of two components: (1) a culture of autologous or homologous bone marrow stem cells partially or completely differentiated into cell lines of a specific connective tissue or (2) extracellular matrix, free from other cellular components secreted by specific connective tissue and the second component is an independent bio-acceptable and biodegradable matrix containing gi esters luronovoy acid. A specific cell line is selected from fibroblasts, osteoblasts, myoblasts, adipocytes, chondrocytes and endothelial cells. This material was previously seeded into keratinocytes prior to the formation of an implant of cellular elements. Cells are cultured in a suitable medium and subsequently these cells can be used immediately or stored for 3 years or more, depending on the degree of cryopreservation, and then already introduced into the carrier. The gel is placed with the grown cells in a bone defect. The implantable material is suitable for use in skin lesions and for repairing damage to connective tissue. However, the described implantation material does not have a sufficiently high biological activity for the restoration of the musculoskeletal system, cannot be considered favorable for the healing process, since the methods for its preparation are standardized for autologous and homologous bone marrow cells and do not take into account the specifics of their differentiation. The limiting conditions include the fact that hyaluronic acid esters are used as the matrix, suggesting a prolonged release of the active components as a result of its degradation and the need to model various forms of the implant.

The analysis of the prior art showed that none of the known technical solutions provides sufficient restoration of tissue of the bone, muscle, cartilage and tendons of the patient using only autologous bone marrow cells of the patient himself, which completely eliminates the risk of antigenicity of the transplanted tissue and pathogenic transformation.

Thus, the aim of the invention was to develop a method for producing an implant for restoration of bone and cartilage tissue, including the study and selection of special conditions and cultivation regimes dictated by the specificity of bone marrow cells, especially elderly patients, for the formation of osteocytes and chondrocytes while maintaining high activity and absolute immune inertness.

Another objective of the invention is the creation of an implant having a higher susceptibility to the patient’s body, minimal integration time compared with the prior art. In other words, the aim of the invention was the creation of such an implant that provides the optimal course of osteochondrogenesis in necrotic bones and degeneratively changed cartilage, providing anti-inflammatory effect, biocompatibility and increasing patient comfort. With regard to the implant, this means that it must quickly and firmly grow together with autogenous tissue, especially bone.

As a result of a large amount of experimental work devoted to the cultivation of stromal fibroblasts of the bone marrow of a patient in need of treatment, we surprisingly found that stromal fibroblasts of the bone marrow, and especially the elderly, require specific conditions for their cultivation, suggesting features of the composition and concentration of the nutrient solution, the modes cultivation, and a certain sequence of their stages. The studies conducted allowed us to select the optimal conditions for the growth of fibroblast cells, to establish those combinations of biologically active substances that allow the cells of patients, including the elderly, to actively proliferate to the desired number. It was established that the achievement of the therapeutic effect is possible already starting from 100,000 and up to 100 million cells. The decisive moment for compliance with these conditions is that the implant according to the invention after the specialization of fibroblasts in the direction of osteocytes or chondrocytes is stored and introduced into the lesion site together with a special nutrient medium corresponding to the conditions for culturing fibroblasts to obtain osteocytes or chondrocytes. For the first time, it was revealed that the combination of specialized fibroblasts obtained in combination with a special nutrient medium ensures the growth and further differentiation of necessary cells and tissues, including in an elderly, ischemic organ, when introduced to the patient.

Thus, one aspect of the invention is an implant for the restoration of bone and / or cartilage, which is osteocytes and / or chondrocytes in a nutrient medium, while osteocytes and / or chondrocytes are obtained from fibroblasts of autologous bone marrow by culturing in a nutrient medium including serum blood of calves, collagen and growth factors, and, optionally, mucopolysaccharides, while the content of osteocytes and / or chondrocytes in the implant is from 100,000 to 100 million cells in 2-5 ml of culture medium, The appropriate fibroblast culture conditions for osteocytes or chondrocytes.

In this case, the implant is intended for insertion into the joint cavity of a bone, tendon, muscle or fascia.

Another aspect of the invention is a method for producing an implant for restoration of bone and / or cartilage tissue, which consists in homogenizing the patient’s bone marrow, then centrifuging, and the sediment containing stromal fibroblasts is cultured in a nutrient medium, while fibroblasts are specialized for fibroblasts towards osteocytes mainly cultivated on a nutrient medium containing in 1 ml of blood serum of calves in an amount of 42.5 to 85 mg, type I collagen in a concentration of 100-350 μg / ml, type II collagen at the end ntration from 10 μg / ml to 100 μg / ml, fibroblast growth factor (FGF) in a concentration of from 0.1 to 0.9 μg / ml; to specialize fibroblasts, mainly towards chondrocytes, fibroblasts are cultured on a nutrient medium, 1 ml of which contains from 42.5 mg to 85 mg of serum, type I and type II collagen in a concentration of 1 to 10 μg / ml each, as well as mucopolysaccharides in concentration from 10 μg / ml to 100 μg / ml each and insulin-like growth factor (IGF-1) or somatomedin hormone in a concentration of from 1.0 to 10.0 μg / ml, cultivation is carried out before accumulation in the nutrient medium from 100,000 to 100 million osteocytes and / or chondrocytes, then 2-5 ml of culture medium are added corresponding to the conditions for culturing fibroblasts to obtain osteocytes or chondrocytes.

Moreover, the method can provide the accumulation of osteocytes up to 50 million in 2 ml of culture medium, and chondrocytes up to 50 million in 5 ml of culture medium.

In this case, the serum is a dry standard deproteinized homogenate from the blood of calves.

The study included 40 patients suffering from various rheumatological diseases complicated by avascular (aseptic necrosis) of the heads of the hip bones and knee joints. The control group consisted of 20 people, 8 of whom had rheumatoid arthritis, 2 with systemic lupus erythematosus, and 10 with osteoarthritis. The second group of patients treated according to this invention also consisted of 20 people: 8 patients suffering from rheumatoid arthritis, two with systemic lupus erythematosus, and 10 people with osteoarthritis aged 16 to 78 years. The duration of the disease ranged from 2 to 15 years. The generally accepted treatment - steroids, vascular therapy, laser joint damage, exercise therapy for patients in the control group was carried out in the surgical department of the Institute of Rheumatology RAMS. In patients who underwent treatment according to the claimed invention, after a diagnosis was established on an outpatient basis, the left iliac wing was punctured and 2 to 6 ml of bone marrow were taken. Then the stromal fibroblasts of the bone marrow of the patient were cultured as described in the method of the invention, an implant was obtained to restore bone and / or cartilage tissue, which contains from 100,000 to 100 million cells in 2-5 ml of culture medium corresponding to the conditions for culturing fibroblasts to obtain osteocytes or chondrocytes. The latter was placed in sterile disposable syringes, or they were combined and placed in a sterile disposable syringe for insertion into the joint cavity, bone, tendon, muscle or fascia. The condition of the patients was evaluated before and after treatment and after 6 months. The visual analogue scale determined the intensity of pain at rest, the amplitude of movements was determined using a goniometer. After the treatment, the condition of the patients improved both in the control group and in the group that underwent implantation with the claimed agent. In the group of patients subjected to cell therapy with an implant, the pain syndrome decreased by 73%, while in the control group by 61%, and goniometry significantly improved by 49% in the group subjected to cell therapy and by 19.7% in the control. However, 6 months after treatment in 18 patients of the control group, all symptoms of aseptic necrosis returned to baseline. Two patients underwent joint replacement surgery. In the group of patients who underwent therapy with the claimed implant, 6 months after treatment in 19 patients, the pain continued to decrease, and the amplitude of movements in the affected joints continued to increase. In 1 patient with rheumatoid arthritis, symptoms of coxitis occurred, after examination, an infection of the genitourinary tract was detected. After the appointment of antibiotics, the condition improved.

Studies have shown that the implant created according to the invention ensures the effectiveness of treatment due to the optimal course of reparative osteochondrogenesis in necrotic bone structures, degeneratively changed cartilage, damaged tendons and muscles.

The effectiveness of treatment, predetermined by the biocompatibility of the implant, was also ensured by the ease of its administration while maintaining high activity and absolute immune inertness.

The implant in all 20 patients of different age groups showed a minimum integration time, high susceptibility of the patient’s body, anti-inflammatory effect, quickly and firmly fused with autogenic tissue, especially bone, and thereby contributed to increasing patient comfort.

The invention is further illustrated in the preferred embodiments of the specific implementation, while the examples of culturing stromal fibroblasts of patients for subsequent autotransplantation into the affected bones, joints, muscles, tendons, and the method of obtaining implants should not become the basis for limiting claims, but are intended solely to demonstrate feasibility inventions and implementation of these purposes.

Example 1

After puncture of a bone affected by aseptic necrosis, the patient is harvested from 2 to 6 ml of his bone marrow. Then, 15 ml of chilled (+ 4 ° C) sterile saline is added to the bone marrow. At the same time, with a syringe through a 0.8 × 38 needle, homogenization is performed. The resulting homogenate is transferred into sterile centrifuge tubes containing approximately 5-fold volume of chilled (+ 4 ° C) sterile saline. The cell suspension is centrifuged for 5 min at 1000 rpm, the supernatant is drained. The pellet containing stromal fibroblasts is resuspended with Eagle's nutrient medium with the usual additives of 1 ml in each tube. Cells in the amount of 2500-10000 are divided into parts for their further specialization.

One part of the cells in an amount of about 1250 to 5000 is sown in Petri dishes to specialize fibroblasts mainly in the direction of osteocytes. At the same time, fibroblasts are cultured on a nutrient medium containing 1 ml of calf blood serum in an amount of 42.5 to 85 mg, type I collagen weighing 25,000-50000 daltons at a concentration of 100-350 μg / ml, type II collagen mol 25,000-50000 dalton in a concentration of 10 μg / ml to 100 μg / ml, fibroblast growth factor (FGF) in a concentration of 0.1 to 0.9 μg / ml.

Some of the cells in an amount of about 1250 to 5000 are sown in Petri dishes to specialize fibroblasts mainly in the direction of chondrocytes. For this, fibroblasts are cultured on a nutrient medium, in 1 ml of which contains from 42.5 mg to 85 mg of serum, collagen type I and type II mol mol. weighing 25000-50000 daltons in a concentration of from 1 to 10 μg / ml each, as well as mucopolysaccharides, preferably chondroitin-4 sulfate and chondroitin-6 sulfate, in a concentration of 10 μg / ml to 100 μg / ml each and a growth factor, preferably insulin-like growth factor (IGF-1) or the hormone somatomedin in a concentration of from 1.0 to 10.0 μg / ml.

Cultivation is carried out before accumulation in the nutrient medium from 100,000 to 100 million specialized cells under ordinary conditions, i.e. laminar cabinets, at approximately 37 ° C in an atmosphere of 5% CO ₂ . On approximately 21 and 35 days of cultivation, cells in each part of the monolayer fibroblast culture are washed with a neutral pH buffer solution, preferably phosphate. After the last dissociation of the cells, they are suspended in a nutrient medium and placed in sterile centrifuge tubes, then the supernatant is separated (possibly by centrifugation for 5 min at 1000 rpm). The supernatant is drained, and 2-5 ml of a special nutrient medium corresponding to the conditions for culturing fibroblasts to obtain osteocytes or chondrocytes is added to the sediment containing cells. The prepared implants are placed in sterile disposable syringes, or they are combined and placed in a sterile disposable syringe.

Example 2

Patient Schmidt T.M. 65 years old, since 2000 suffers from pains in the area of the right hip joint. In January 2004 she was hospitalized in the surgical department of the Institute of Rheumatology of the Russian Academy of Medical Sciences, where she was diagnosed with arthrosis of the hip joints, art. III-IV, aseptic necrosis of the head of the right femur. Recommended surgery - joint replacement.

On an outpatient basis, she underwent novocaine analgesia for an extraverticular puncture of the right femur. The needle was inserted towards the center of the head, to a depth of 5 cm. Then the bone marrow was taken in an amount of 4 ml, its stromal fibroblasts were cultured for 32 days according to the above procedure to 100 million. Then, 50 million fibroblasts specialized in the direction osteocytes, together with 2 ml of a special nutrient solution were injected heart-worm into the neck of the right femur. 50 million fibroblasts specialized in chondrocytes, together with 5 ml of a special nutrient medium, were introduced into an ultrasound-guided center of the right hip joint under ultrasound control, followed by passive-active movements in this joint.

As a result of cell therapy with specialized fibroblasts of the affected joint and bone in a patient 14 days after autotransplantation, functional tests began to significantly improve and pain syndrome decreased. So, the pain during movement from 10 cm (according to YOUR) decreased to 5 cm, and after one month, it completely disappeared. The function of the right hip joint (according to Harris) before treatment was 41 points, after 14 days - 64, after a month 73 points. And what is most remarkable, an external rotation appeared in the joint (0-10-25 degrees), which, in principle, does not happen with such a pathology.

Example 3

Patient Mozhaeva V.V. 35 years old, complained of severe pain and lameness in the area of the right hip joint. Considers herself a patient since 1998, when, for no apparent reason, pain appeared in the right inguinal region. In February 2004 she was hospitalized at the Institute of Rheumatology of the Russian Academy of Medical Sciences, where she was diagnosed with dysplastic arthrosis of the hip joints, art. III-IV, aseptic necrosis of the head of the right femur. Right-sided trochanteritis. After which she was recommended surgery to replace the joint with an endoprosthesis.

On an outpatient basis, she underwent transocentential puncture of the right femur under novocaine analgesia. The needle was inserted towards the center of the head, to a depth of 5 cm. Then, the bone marrow was taken in an amount of 3 ml for 17 days, its stromal fibroblasts were cultured according to the above procedure to 100 million. Then, 50 million cells specialized in osteocytes, Together with 2 ml of special nutrient medium, they were injected heart-wormly into the neck of the right femur. 50 million cells specialized in the direction of chondrocytes, together with 5 ml of a special nutrient medium, were introduced under ultrasound control into the center of the joint cavity of the right hip joint, followed by passive-active movements in this joint. And 500,000 cells that underwent specialization in the direction of chondrocytes were introduced together with 5 ml of culture medium into the area of attachment of the tendon of the broadest fascia of the right thigh to the greater trochanter.

As a result of cell therapy with specialized fibroblasts of the affected joint and bone in the patient, 14 days after autotransplantation, functional tests began to significantly improve and pain syndrome decreased. So the pain during movement from 9 cm (according to YOUR) decreased to 3 cm, and after one month periodically arose with a force of 2-3 cm. The function of the right hip joint (according to Harris) before treatment was 26 points, after 14 days - 65, through month 75 points. External rotation increased from 15 to 20 degrees. The signs of right-sided trochanteritis and symptoms of tendon inflammation of the broadest fascia of the thigh disappeared.

Example 4

Patient Dubrovina M.M. 66 years old, complained of severe pain in the knee joints, more on the left, inability to walk for a long distance. In February 2004, she was hospitalized at the Institute of Rheumatology, Russian Academy of Medical Sciences, where she was diagnosed with arthrosis of the knee joints, art. III-IV, aseptic necrosis of the left knee joint, tendonitis and myositis of the distal tailor muscle on the left, according to the destructive type. An operation to replace the joint with an endoprosthesis was recommended.

On an outpatient basis, she underwent a puncture metaphysis of the left tibia to a depth of 4 cm under procaine anesthesia and a bone marrow of 4 ml was taken. For 26 days, its stromal fibroblasts were cultured according to the aforementioned method to 100 million. Then, 50 million cells specialized in the direction of osteocytes, together with 4 ml of special nutrient medium, were alternately introduced (2 ml + 50 million each) into the medial condyle of the left femoral and of the left tibia, where according to NMR the largest destructive changes were detected. 40 million cells that passed, specialization in the direction of chondrocytes, together with 5 ml of a special nutrient medium, were introduced into the cavity of the left knee joint, between the medial and lateral condyles of the femur and tibia. 10 million cells specializing in the direction of chondrocytes were introduced 5 ml each into the tendon and distal section of the tailor muscle on the left, respectively.

14 days after autotransplantation in a patient suffering from overweight (106 kg), functional tests began to significantly improve and pain was reduced. The pain during movement decreased from 7 cm (according to YOUR) to 4 cm and after one month was 3 cm. Function in the left knee joint increased from 25 to 43 points.

Example 5

Prescribed epicrisis from medical history No. 3004C / 2003. Inessa Sergeevna, 74, was examined and treated at the orthopedic and surgical department of the Clinic of the Institute of Rheumatology, Russian Academy of Medical Sciences, from 06.10.03 to 10.24.2003 (18 beds) with a diagnosis of Polyostearthrosis , arthrosis of the shoulder joints st. 1-2. Left-side humeroscapular periarthritis (tendonitis of the subscapular and 2-headed muscles of the left).

Received with complaints of severe pain in the cervical, lumbar spine, shoulder joints. Limited function in the left shoulder joint. Considers herself a patient since 1986, when pain in the cervical spine and left shoulder joint first appeared. Gradually, the disease progressed. Locally: on palpation, painful points are determined in the area of the spinous processes of the spine, shoulder joints, long head of the tendon with a 2-head, subscapular and deltoid muscles on the left.

The range of motion in them is limited, more in the left shoulder joint.

Ultrasound of the shoulder joints: narrowing of the joint spaces in the acromioclavicular joints. Reducing the thickness of the tendons of the 2-head and subscapularis muscle on the left. Ultrasound - a picture of left-sided tendonitis of the infraspinatus and 2-headed muscles, degenerative-dystrophic changes in the tendons of the subscapular and 2-headed muscles.

Conducted conservative therapy did not bring any effect. After discharge from the hospital, the patient underwent cell therapy with their own stem cells, according to the above scheme. So, 50 million cells specialized in the direction of chondrocytes were used as follows. 25 million cells were introduced into the area of the thinned subscapular muscle tendon; in the tendon of the long head of the 2-headed muscle of 25 million cells.

After 6 months after cell therapy, the patient noted a significant improvement in well-being, pain in the left shoulder joint decreased by 75%, and the amplitude of movement (flexion) increased by 80 degrees.

Claims (6)

1. An implant for the restoration of bone and / or cartilage, characterized in that it is osteocytes and / or chondrocytes in a nutrient medium, while the osteocytes and / or chondrocytes are obtained from fibroblasts of autologous bone marrow by cultivation in a nutrient medium including blood serum calves, collagen and growth factors, and, optionally, mucopolysaccharides, while the content of osteocytes and / or chondrocytes in the implant is from 100,000 to 100 million cells in 2-5 ml of culture medium that meets the conditions of tivirovaniya fibroblasts for osteocytes or chondrocytes. 2. The implant according to claim 1, which is intended for insertion into the joint cavity, bone, tendon, muscle or fascia. 3. A method of obtaining an implant for restoration of bone and / or cartilage tissue, characterized in that the patient’s bone marrow is homogenized, then centrifuged, the precipitate containing stromal fibroblasts is cultured on a nutrient medium, while for the specialization of fibroblasts towards osteocytes, fibroblasts are mainly cultivated on nutrient medium containing 1 ml of blood serum of calves in an amount of 42.5 to 85 mg, collagen type I at a concentration of 100-350 μg / ml, collagen type II at a concentration of 10 to 100 μg / ml, growth factor Ibroblasts (FGF) in a concentration of 0.1 to 0.9 μg / ml, for the specialization of fibroblasts mainly in the direction of chondrocytes, fibroblasts are cultured on a nutrient medium, in 1 ml of which contains from 42.5 to 85 mg of serum, type I and type collagen II at a concentration of 1 to 10 μg / ml each, as well as mucopolysaccharides at a concentration of 10 to 100 μg / ml each and insulin-like growth factor (IGF-1) or somatomedin hormone at a concentration of 1.0 to 10.0 μg / ml , cultivation is carried out before accumulation in a nutrient medium from 100,000 to 100 million cells, then 2-5 ml of production medium corresponding to the conditions for the cultivation of fibroblasts to obtain osteocytes or chondrocytes. 4. The method according to claim 3, which provides for the accumulation of osteocytes up to 50 million in 2 ml of culture medium. 5. The method according to claim 3, which provides for the accumulation of chondrocytes up to 50 million in 5 ml of culture medium. 6. The method according to claim 3, in which the serum is a dry standard deproteinized homogenate from the blood of calves.